大肠杆菌膦酸酯代谢途径中phnF基因的研究

Studies on phn F in the Phosphonate Utilization Pathway of Escherichia coli

大肠杆菌的ｐｈｎ操纵子与膦酸酯（Ｐｎ）的利用密切相关。实验利用ＰＣＲ扩增、ＴＡ克隆等方法，获得了大肠杆菌ｐｈｎ操纵子中ｐｈｎＥ、ｐｈｎＦ和ｐｈｎＧ基因的亚克隆，并进行了序列测定。通过Ｐ１噬菌体转导，构建了ｐｈｎＦ的ＴｎｐｈｏＡ’９转座子插入突变体ＪＷ１９，该突变仅对大肠杆菌的ＡＥＰｎ同化有微弱的影响，而利用ｐｈｎＥ和ｐｈｎＧ序列与染色体重组构建的ｐｈｎＦ全缺失突变株ＪＷ６７，则几乎不能在ＡＥＰｎ培养基上生长。通过ｐｈｎＦ基因的诱导高表达，用亲和柱层析分离纯化了ＰｈｎＦ蛋白，达到电泳纯。并且用凝胶延滞的方法观察到，ＰｈｎＦ蛋白与ｐｈｎ操纵子ＤＮＡ片段相互作用后，可使凝胶图谱类型发生改变。

The phn operon is responsible for phosphonate utilization of E.coli . phn E, phn F and phn G genes were cloned by PCR and TA cloning method,and their nucleotide sequences have been determined.By P1 phage transduction,the mutant of phn F∷TnphoA′ 9(JW19) was constructed.The phn F clean deletion in JW67 chromosome was also constructed by allele replacement of phn E and phn G sequences.According to their growth on AEPn MOPS medium,TnphoA′ 9 only has a mild effect on Pn utilization of JW19 while JW67 nearly lost the ability to use Pn as P source.PhnF protein was overexpressed and purified by affinity chromatography.The purified PhnF protein was used to interact with the entire phn operon.Gel reatardation experiment showed an obvious change on the pattern of gel bands.Thus we learned that PhnF protein is probably a regulatory protein,which acts through binding to DNA.